Auto- and cross-regulation of the hnRNPs D and DL.

HnRNP D, better known as AUF1, is an extensively studied protein that controls a variety of cellular pathways. Consequently, its expression has to be tightly regulated to prevent the onset of pathologies. In contrast, the cellular functions and regulation of its ubiquitously expressed paralog hnRNP DL are barely explored. Here, we present an intricate crosstalk between these two proteins. Both hnRNP D and DL are able to control their own expression by alternative splicing of cassette exons in their 3'UTRs. Exon inclusion produces mRNAs degraded by nonsense-mediated decay. Moreover, hnRNP D and DL control the expression of one another by the same mechanism. Thus, we identified two novel ways of how hnRNP D expression is controlled. The tight interconnection of expression control directly links hnRNP DL to hnRNP D-related diseases and emphasizes the importance of a systematic analysis of its cellular functions.

AUF1 modulates TGF-ß signal in renal tubular epithelial cells via post-transcriptional regulation of Nedd4L expression.

Posttranscriptional regulation process plays important roles in renal disease pathogenesis. AU-rich element RNA-binding protein (AUF1) interacts with and destabilizes mRNAs containing AU-rich elements (AREs) in their 3'UTR. The current study demonstrated that AUF1 was increased in unilateral ureteral obstruction (UUO) animal models. While proliferation and migration of HK2 cells was unaltered by AUF1 downregulation under normal condition, proliferative inhibition and migratory promotion mediated by TGF-ß was significantly compromised. Mechanically, AUF1 downregulation decreased phosphorylated Smad2/3 via increasing their E3 ligase Nedd4L at the posttranscriptional level. In addition, the current study identified Nedd4L as a previously unreported target of AUF1. AUF1 regulates Nedd4L expression at the posttranscriptional level by interaction with AREs in the 3'UTR of the Nedd4L mRNA. Collectively, the current study indicates that AUF1 might be a potential player in renal tubulointerstitial fibrosis through modulation of TGF-ß signal transduction via posttranscriptional regulation of Nedd4L.

Energy stress-induced lncRNA FILNC1 represses c-Myc-mediated energy metabolism and inhibits renal tumor development.

The roles of long non-coding RNAs in cancer metabolism remain largely unexplored. Here we identify FILNC1 (FoxO-induced long non-coding RNA 1) as an energy stress-induced long non-coding RNA by FoxO transcription factors. FILNC1 deficiency in renal cancer cells alleviates energy stress-induced apoptosis and markedly promotes renal tumor development. We show that FILNC1 deficiency leads to enhanced glucose uptake and lactate production through upregulation of c-Myc. Upon energy stress, FILNC1 interacts with AUF1, a c-Myc mRNA-binding protein, and sequesters AUF1 from binding c-Myc mRNA, leading to downregulation of c-Myc protein. FILNC1 is specifically expressed in kidney, and is downregulated in renal cell carcinoma; also, its low expression correlates with poor clinical outcomes in renal cell carcinoma. Together, our study not only identifies FILNC1 as a negative regulator of renal cancer with potential clinical value, but also reveals a regulatory mechanism by long non-coding RNAs to control energy metabolism and tumor development.FoxO are commonly down-regulated transcription factors and tumor suppressors in renal cell cancer (RCC). Here, the authors show that upon energy stress FoxOs induce the expression of the long non-coding RNA FILNC1, which inhibits survival of RCC by downregulating c-Myc and c-Myc-dependent metabolic rewiring.

Induction of epithelial-mesenchymal transition (EMT) by Beclin 1 knockdown via posttranscriptional upregulation of ZEB1 in thyroid cancer cells.

Beclin 1 has emerged as a haploinsufficient tumor suppression gene in a variety of human carcinomas. In order to clarify the role of Beclin 1 in thyroid cancer, Beclin 1 was knockdown in thyroid cancer cell lines. The current study demonstrated that knockdown of Beclin 1 resulted in morphological and molecular changes of thyroid cancer cells consistent with epithelial-mesenchymal transition (EMT), a morphogenetic procedure during which cells lose their epithelial characteristics and acquire mesenchymal properties concomitantly with gene expression reprogramming. In addition, the current study presented evidence demonstrating that Beclin 1 knockdown triggered this prometastatic process via stabilization of the EMT inducer ZEB1 mRNA through upregulation of AU-binding factor 1 (AUF1), which is recruited to the 3'-untranslated region (UTR) of the ZEB1 mRNA and decreases its degradation. We also found a negative correlation of Beclin 1 with AUF1 or ZEB1 in thyroid cancer tissues. These results indicated that at least some tumor suppressor functions of Beclin 1 were mediated through posttranscriptional regulation of ZEB1 via AUF1 in thyroid cancers.

The superoxide dismutase 1 3'UTR maintains high expression of the SOD1 gene in cancer cells: The involvement of the RNA-binding protein AUF-1.

Superoxide dismutase 1 (SOD1) is ubiquitously expressed and the predominant dismutase in the cytoplasm. Whereas transcriptional regulation of the SOD1 gene has been well characterized, posttranscriptional regulation of the gene remains largely unknown in eukaryotes. In this study, a full-length 3'UTR of the SOD1 transcript was cloned and characterized for its ability to regulate SOD1 gene expression in human cancer cells. Inclusion of the SOD1 3'UTR in the pGL3 reporter construct dramatically enhanced the reporter activity by 10- to 220-fold in various cell lines. RT-PCR analysis, however, indicated that the reporter gene mRNA levels were only modestly altered by the SOD1 3'UTR, suggesting that the SOD1 3'UTR enhances the reporter gene activity not simply by stabilizing the mRNA but primarily by promoting translation of the protein. Bioinformatics analysis showed multiple stem and loop structures of the SOD1 3'UTR, and alterations in this secondary structure led to remarkably reduced reporter gene activity. Importantly, introducing the SOD1 3'UTR into cancer cells attenuated endogenous SOD1 expression in a concentration-dependent manner, indicating the involvement of RNA trans-acting factors in this process. Using siRNA and RNA immunoprecipitation techniques, we identified AUF-1, an RNA-binding protein, as a positive regulator of SOD1 expression through its 3'UTR. Consequently, AUF-1 was found to regulate redox balance in our cell model systems. Furthermore, in human ovarian, esophageal, and pancreatic cancer tissues, the expression of SOD1 was significantly correlated with that of AUF-1, further supporting the importance of AUF-1 in regulating SOD1 gene expression.

Role of Auf1 in elimination of oxidatively damaged messenger RNA in human cells.

In aerobically growing cells, in which reactive oxygen species are produced, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine, which induces alterations in gene expression. Here we show that the human Auf1 protein, also called HNRNPD, binds specifically to RNA containing this oxidized base and may be involved in cellular processes associated with managing the problems caused by RNA oxidation. Auf1-deficient cells were constructed from human HeLa and Nalm-6 lines using two different targeting procedures. Both types of Auf1-deficient cells are viable, but exhibit growth retardation. The stability of messenger RNA for four different housekeeping genes was determined in Auf1-deficient and -proficient cells, treated with or without hydrogen peroxide. The level of oxidized messenger RNA was considerably higher in Auf1-deficient cells than in Auf1-proficient cells. Auf1 may play a role in the elimination of oxidized RNA, which is required for the maintenance of proper gene expression under conditions of oxidative stress.

MicroRNA-141 and microRNA-146b-5p inhibit the prometastatic mesenchymal characteristics through the RNA-binding protein AUF1 targeting the transcription factor ZEB1 and the protein kinase AKT.

miR-141 and miR-146b-5p are two important tumor suppressor microRNAs, which control several cancer-related genes and processes. In the present report, we have shown that these microRNAs bind specific sites at the 3'-untranslated region (UTR) of the mRNA-binding protein AUF1, leading to its down-regulation. This inverse correlation between the levels of these microRNAs and AUF1 has been identified in various osteosarcoma cell lines. Additionally, we present clear evidence that AUF1 promotes mesenchymal features in osteosarcoma cells and that miR-141 and miR-146b-5p suppress this prometastatic process through AUF1 repression. Indeed, both microRNAs suppressed the invasion/migration and proliferation abilities of osteosarcoma cells through inhibiting the AKT protein kinase in an AUF1-dependent manner. We have also shown that AUF1 binds to and stabilizes the mRNA of the AKT activator phosphoinositide-dependent kinase-1 (PDK1). Furthermore, miR-141 and miR-146b-5p positively regulate the epithelial markers (E-cadherin and Epcam) and repress the mesenchymal markers (N-cadherin, Vimentin, Twist2, and ZEB1). These effects were mediated via the repression of the epithelial-to-mesenchymal inducer ZEB1 through targeting AUF1, which binds the 3'-UTR of the ZEB1 mRNA and reduces its turnover. These results indicate that at least some tumor suppressor functions of miR-141 and miR-146b-5p are mediated through the repression of the oncogenic potentials of AUF1. Therefore, these 3'-UTR-directed post-transcriptional gene expression regulators constitute promising new targets for diagnostic and/or therapeutic interventions.

RNA-binding protein AUF1 promotes myogenesis by regulating MEF2C expression levels.

The mammalian RNA-binding protein AUF1 (AU-binding factor 1, also known as heterogeneous nuclear ribonucleoprotein D [hnRNP D]) binds to numerous mRNAs and influences their posttranscriptional fate. Given that many AUF1 target mRNAs encode muscle-specific factors, we investigated the function of AUF1 in skeletal muscle differentiation. In mouse C2C12 myocytes, where AUF1 levels rise at the onset of myogenesis and remain elevated throughout myocyte differentiation into myotubes, RNP immunoprecipitation (RIP) analysis indicated that AUF1 binds prominently to Mef2c (myocyte enhancer factor 2c) mRNA, which encodes the key myogenic transcription factor MEF2C. By performing mRNA half-life measurements and polysome distribution analysis, we found that AUF1 associated with the 3' untranslated region (UTR) of Mef2c mRNA and promoted MEF2C translation without affecting Mef2c mRNA stability. In addition, AUF1 promoted Mef2c gene transcription via a lesser-known role of AUF1 in transcriptional regulation. Importantly, lowering AUF1 delayed myogenesis, while ectopically restoring MEF2C expression levels partially rescued the impairment of myogenesis seen after reducing AUF1 levels. We propose that MEF2C is a key effector of the myogenesis program promoted by AUF1.

HuR and other turnover- and translation-regulatory RNA-binding proteins: implications for the kidney.

The posttranscriptional regulation of gene expression occurs through cis RNA regulatory elements by the action of trans factors, which are represented by noncoding RNAs (especially microRNAs) and turnover- and translation-regulatory (TTR) RNA-binding proteins (RBPs). These multifactorial proteins are a group of heterogeneous RBPs primarily implicated in controlling the decay and translation rates of target mRNAs. TTR-RBPs usually shuttle between cellular compartments (the nucleus and cytoplasm) in response to various stimuli and undergo posttranslational modifications such as phosphorylation or methylation to ensure their proper subcellular localization and function. TTR-RBPs are emerging as key regulators of a wide variety of genes influencing kidney physiology and pathology. This review summarizes the current knowledge of TTR-RBPs that influence renal metabolism. We will discuss the role of TTR-RBPs as regulators of kidney ischemia, fibrosis and matrix remodeling, angiogenesis, membrane transport, immunity, vascular tone, hypertension, and acid-base balance as well as anemia, bone mineral disease, and vascular calcification.

Cellular mRNA decay protein AUF1 negatively regulates enterovirus and human rhinovirus infections.

To successfully complete their replication cycles, picornaviruses modify several host proteins to alter the cellular environment to favor virus production. One such target of viral proteinase cleavage is AU-rich binding factor 1 (AUF1), a cellular protein that binds to AU-rich elements, or AREs, in the 3' noncoding regions (NCRs) of mRNAs to affect the stability of the RNA. Previous studies found that, during poliovirus or human rhinovirus infection, AUF1 is cleaved by the viral proteinase 3CD and that AUF1 can interact with the long 5' NCR of these viruses in vitro. Here, we expand on these initial findings to demonstrate that all four isoforms of AUF1 bind directly to stem-loop IV of the poliovirus 5' NCR, an interaction that is inhibited through proteolytic cleavage of AUF1 by the viral proteinase 3CD. Endogenous AUF1 was observed to relocalize to the cytoplasm of infected cells in a viral protein 2A-driven manner and to partially colocalize with the viral protein 3CD. We identify a negative role for AUF1 in poliovirus infection, as AUF1 inhibited viral translation and, ultimately, overall viral titers. Our findings also demonstrate that AUF1 functions as an antiviral factor during infection by coxsackievirus or human rhinovirus, suggesting a common mechanism that targets these related picornaviruses.

Association of the von Hippel-Lindau protein with AUF1 and posttranscriptional regulation of VEGFA mRNA.

The von Hippel-Lindau (VHL) tumor suppressor gene product is the recognition component of an E3 ubiquitin ligase and is inactivated in patients with VHL disease and in most sporadic clear-cell renal cell carcinomas (RCC). pVHL controls oxygen-responsive gene expression at the transcriptional and posttranscriptional levels. The VEGFA mRNA contains AU-rich elements (ARE) in the 3'-untranslated region, and mRNA stability or decay is determined through ARE-associated RNA-binding factors. We show here that levels of the ARE-binding factor, AUF1, are regulated by pVHL and by hypoxia. pVHL and AUF1 stably associate with each other in cells and AUF1 is a ubiquitylation target of pVHL. AUF1 and another RNA-binding protein, HuR, bind to VEGFA ARE RNA. Ribonucleoprotein (RNP) immunoprecipitations showed that pVHL associates indirectly with VEGFA mRNA through AUF1 and/or HuR, and this complex is associated with VEGFA mRNA decay under normoxic conditions. Under hypoxic conditions pVHL is downregulated, whereas AUF1 and HuR binding to VEGF mRNA is maintained, and this complex is associated with stabilized mRNA. These studies suggest that AUF1 and HuR bind to VEGFA ARE RNA under both normoxic and hypoxic conditions, and that a pVHL-RNP complex determines VEGFA mRNA decay. These studies further implicate the ubiquitin-proteasome system in ARE-mediated RNA degradation.

Differential effects of hnRNP D/AUF1 isoforms on HIV-1 gene expression.

Control of RNA processing plays a major role in HIV-1 gene expression. To explore the role of several hnRNP proteins in this process, we carried out a siRNA screen to examine the effect of depletion of hnRNPs A1, A2, D, H, I and K on HIV-1 gene expression. While loss of hnRNPs H, I or K had little effect, depletion of A1 and A2 increased expression of viral structural proteins. In contrast, reduced hnRNP D expression decreased synthesis of HIV-1 Gag and Env. Loss of hnRNP D induced no changes in viral RNA abundance but reduced the accumulation of HIV-1 unspliced and singly spliced RNAs in the cytoplasm. Subsequent analyses determined that hnRNP D underwent relocalization to the cytoplasm upon HIV-1 infection and was associated with Gag protein. Screening of the four isoforms of hnRNP D determined that, upon overexpression, they had differential effects on HIV-1 Gag expression, p45 and p42 isoforms increased viral Gag synthesis while p40 and p37 suppressed it. The differential effect of hnRNP D isoforms on HIV-1 expression suggests that their relative abundance could contribute to the permissiveness of cell types to replicate the virus, a hypothesis subsequently confirmed by selective depletion of p45 and p42.

AU-rich RNA binding proteins in hematopoiesis and leukemogenesis.

Posttranscriptional mechanisms are now widely acknowledged to play a central role in orchestrating gene-regulatory networks in hematopoietic cell growth, differentiation, and tumorigenesis. Although much attention has focused on microRNAs as regulators of mRNA stability/translation, recent data have highlighted the role of several diverse classes of AU-rich RNA-binding protein in the regulation of mRNA decay/stabilization. AU-rich elements are found in the 3'-untranslated region of many mRNAs that encode regulators of cell growth and survival, such as cytokines and onco/tumor-suppressor proteins. These are targeted by a burgeoning number of different RNA-binding proteins. Three distinct types of AU-rich RNA binding protein (ARE poly-U-binding degradation factor-1/AUF1, Hu antigen/HuR/HuA/ELAVL1, and the tristetraprolin/ZFP36 family of proteins) are essential for normal hematopoiesis. Together with 2 further AU-rich RNA-binding proteins, nucleolin and KHSRP/KSRP, the functions of these proteins are intimately associated with pathways that are dysregulated in various hematopoietic malignancies. Significantly, all of these AU-rich RNA-binding proteins function via an interconnected network that is integrated with microRNA functions. Studies of these diverse types of RNA binding protein are providing novel insight into gene-regulatory mechanisms in hematopoiesis in addition to offering new opportunities for developing mechanism-based targeted therapeutics in leukemia and lymphoma.

Role of AUF1 and HuR in the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK1-F⁺ cells.

Onset of metabolic acidosis leads to a rapid and pronounced increase in expression of phosphoenolpyruvate carboxykinase (PEPCK) in rat renal proximal convoluted tubules. This adaptive response is modeled by treating a clonal line of porcine LLC-PK(1)-F(+) cells with an acidic medium (pH 6.9, 9 mM HCO(3)(-)). Measurement of the half-lives of PEPCK mRNA in cells treated with normal (pH 7.4, 26 mM HCO(3)(-)) and acidic medium established that the observed increase is due in part to stabilization of the PEPCK mRNA. The pH-responsive stabilization was reproduced in a Tet-responsive chimeric reporter mRNA containing the 3'-UTR of PEPCK mRNA. This response was lost by mutation of a highly conserved AU sequence that binds AUF1 and is the primary element that mediates the rapid turnover of PEPCK mRNA. However, siRNA knockdown of AUF1 had little effect on the basal levels and the pH-responsive increases in PEPCK mRNA and protein. Electrophoretic mobility shift assays established that purified recombinant HuR, another AU element binding protein, also binds with high affinity and specificity to multiple sites within the final 92-nucleotides of the 3'-UTR of the PEPCK mRNA, including the highly conserved AU-rich element. siRNA knockdown of HuR caused pronounced decreases in basal expression and the pH-responsive increases in PEPCK mRNA and protein. Therefore, basal expression and the pH-responsive stabilization of PEPCK mRNA in LLC-PK(1)-F(+) cells, and possibly in the renal proximal tubule, may require the remodeling of HuR and AUF1 binding to the elements that mediate the rapid turnover of PEPCK mRNA.

The role of AUF1 in regulated mRNA decay.

Messenger ribonucleic acid (mRNA) turnover is a major control point in gene expression. In mammals, many mRNAs encoding inflammatory cytokines, oncoproteins, and G-protein-coupled receptors are destabilized by the presence of AU-rich elements (AREs) in their 3'-untranslated regions. Association of ARE-binding proteins (AUBPs) with these mRNAs promotes rapid mRNA degradation. ARE/poly(U)-binding/degradation factor 1 (AUF1), one of the best-characterized AUBPs, binds to many ARE-mRNAs and assembles other factors necessary to recruit the mRNA degradation machinery. These factors include translation initiation factor eIF4G, chaperones hsp27 and hsp70, heat-shock cognate protein hsc70, lactate dehydrogenase, poly(A)-binding protein, and other unidentified proteins. Numerous signaling pathways alter the composition of this AUF1 complex of proteins to effect changes in ARE-mRNA degradation rates. This review briefly describes the roles of mRNA decay in gene expression in general and ARE-mediated decay (AMD) in particular, with a focus on AUF1 and the different modes of regulation that govern AUF1 involvement in AMD.

Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1beta mRNA.

In human monocyte-derived macrophages, the MCPIP gene (monocyte chemoattractant protein-induced protein) is strongly activated by interleukin-1beta (IL-1beta). Using bioinformatics, a PIN domain was identified, spanning amino acids 130-280; such domains are known to possess structural features of RNases. Recently, RNase properties of MCPIP were confirmed on transcripts coding for interleukins IL-6 and IL-12p40. Here we present evidence that siRNA-mediated inhibition of the MCPIP gene expression increases the level of the IL-1beta transcript in cells stimulated with LPS, whereas overexpression of MCPIP exerts opposite effects. Cells with an increased level of wild-type MCPIP showed lower levels of IL-1beta mRNA. However, this was not observed when mutant forms of MCPIP, either entirely lacking the PIN domain or with point mutations in this domain, were used. The results of experiments with actinomycin D indicate that lower levels of IL-1beta mRNA are due to shortening of the IL-1beta transcript half-life, and are not related to the presence of AU-rich elements in the 3' UTR. The interaction of the MCPIP with transcripts of both IL-1beta and MCPIP observed in an RNA immunoprecipitation assay suggests that this novel RNase may be involved in the regulation of expression of several genes.

The role of AUF1 in thyroid carcinoma progression.

AUF1/heterogeneous nuclear ribonucleoprotein D is an adenylate-uridylate-rich elements (AREs) -binding protein, which regulates the mRNA stability of many genes related to growth regulation, such as proto-oncogenes, growth factors, cytokines, and cell cycle-regulatory genes. Several studies demonstrated AUF1 involvement in the processes of apoptosis, tumorigenesis, and development by its interactions with ARE-bearing mRNAs. We report here that AUF1 may be involved in thyroid carcinoma progression. Investigations on thyroid tissues revealed that cytoplasmic expression of AUF1 in malignant tissues was increased when compared with benign thyroid tissues. In thyroid carcinoma cell lines, AUF1 was mostly detectable in the nucleus; however, in dividing cells, its increased production was also observed in the cytoplasm. We found AUF1 in complexes with ARE-bearing mRNAs, previously described to be crucial for proliferation and cell cycle of thyroid carcinoma. Total or exon-selective knockdown of AUF1 led to growth inhibition accompanied by induction of cell cycle inhibitors and decreased levels of cell cycle promoters. Our data demonstrate the existence of a complex network between AUF1 and mRNAs encoding proteins related to cell proliferation. AUF1 may control the balance between stabilizing and destabilizing effects, both of which are exerted on cell cycle machinery in thyroid carcinoma. Although we cannot exclude participation of other factors, thyroid carcinoma may recruit cytoplasmic AUF1 to disturb the stability of mRNAs encoding cyclin-dependent kinase inhibitors, leading to uncontrolled growth and progression of tumor cells. Thus, AUF1 may be considered as a new, additional marker for thyroid carcinoma.

Similar regulation of human inducible nitric-oxide synthase expression by different isoforms of the RNA-binding protein AUF1.

The ARE/poly-(U) binding factor 1 (AUF1), a protein family consisting of four isoforms, is believed to mediate mRNA degradation by binding to AU-rich elements (ARE). However, evidence exists that individual AUF1 isoforms may stabilize ARE-containing mRNAs. The 3'-untranslated region of the human inducible nitric-oxide synthase (iNOS) contains five AREs, which promote RNA degradation. We have recently shown that the RNA-binding protein KSRP is critically involved in the decay of the iNOS mRNA. In this study we examined the effects of the individual AUF1 isoforms on iNOS expression. Overexpression of each AUF1 isoform reduces iNOS expression on mRNA and protein levels to the same extent by modulation of mRNA stability. Accordingly, knockdown of all or individual AUF1 isoforms by an RNA interference approach enhances iNOS expression. The AUF1 effect on iNOS expression is dependent on the iNOS 3'-untranslated region sequence, as demonstrated in transfection experiments with a reporter mRNA. Binding studies showed that all AUF1 isoforms interact with the same AU-rich region in the iNOS-3'-untranslated region. Cytokine stimulation altered intracellular AUF1 binding activities. These data demonstrate that AUF1 is an important factor that promotes iNOS mRNA degradation. Furthermore, all individual AUF1 isoforms act in a similar manner.

Auf1/Hnrnpd-deficient mice develop pruritic inflammatory skin disease.

Mice lacking heterogenous nuclear ribonuclear protein D (Hnrnpd), also known as Auf1, a regulator of inflammatory cytokine mRNA stability, develop chronic dermatitis with age that is characterized by pruritus and excoriations. Histological analysis showed marked epidermal acanthosis and spongiosis, neovascularization, and elevated number of inflammatory cells, including T cells, macrophages, neutrophils, mast cells, and eosinophils. Hnrnpd-deficient (Hnrnpd(tm1Rjsc)) mice with dermatitis display elevated serum IgE levels. Lesions in Hnrnpd(tm1Rjsc) mice were associated with a shift towards a Th(2) immune environment. Evaluation of T-cell-mediated skin inflammation by assaying contact hypersensitivity indicated an increased response in Hnrnpd(tm1Rjsc) mice. T cells and macrophages from Hnrnpd(tm1Rjsc) mice demonstrate a number of abnormalities associated with dermatitis, including increased IL2, tumor-necrosis factor-alpha (TNFalpha), and IL1beta production. Finally, many features of spontaneous dermatitis could be recapitulated in experimentally induced lesions by subcutaneous injection of CCL27 and TNF in unaffected Hnrnpd(tm1Rjsc) mice. Collectively, these data highlight the importance of HNRNPD and proper regulation of mRNA stability in the intricate processes of leukocyte recruitment and inflammatory activation within the skin.

v-Src oncogene product increases sphingosine kinase 1 expression through mRNA stabilization: alteration of AU-rich element-binding proteins.

Sphingosine kinase 1 (SPHK1) is overexpressed in solid tumors and leukemia. However, the mechanism of SPHK1 overexpression by oncogenes has not been defined. We found that v-Src-transformed NIH3T3 cells showed a high SPHK1 mRNA, SPHK1 protein and SPHK enzyme activity. siRNA of SPHK1 inhibited the growth of v-Src-NIH3T3, suggesting the involvement of SPHK1 in v-Src-induced oncogenesis. v-Src-NIH3T3 showed activations of protein kinase C-alpha, signal transducers and activators of transcription 3 and c-Jun NH(2)-terminal kinase. Their inhibition suppressed SPHK1 expression in v-Src-NIH3T3, whereas their overexpression increased SPHK1 mRNA in NIH3T3. Unexpectedly, the nuclear run-on assay and the promoter analysis using 5'-promoter region of mouse SPHK1 did not show any significant difference between mock- and v-Src-NIH3T3. Furthermore, the half-life of SPHK1 mRNA in mock-NIH3T3 was nearly 15 min, whereas that of v-Src-NIH3T3 was much longer. Examination of two AU-rich region-binding proteins, AUF1 and HuR, that regulate mRNA decay reciprocally, showed decreased total AUF1 protein associated with increased tyrosine-phosphorylated form and increased serine-phosphorylated HuR protein in v-Src-NIH3T3. Modulation of AUF1 and HuR by their overexpression or siRNA revealed that SPHK1 mRNA in v-Src- and mock-NIH3T3 was regulated reciprocally by these factors. Our results showed, for the first time, a novel mechanism of v-Src-induced SPHK1 overexpression.